Echinocandin lipopeptides are natural products that have shown potent fungicidal activity against Aspergillus and Candida sp. and protect animals from fungicidal infection by inhibiting the enzyme β(1,3)-D-Glucan synthase, thereby disturbing the integrity of the fungal cell wall. Echinocandin family compounds are natural products such as Echinocandin B, Echinocandin C, Mulundocandin, Sporiofungin A, Pneumocandin A0, and Pneumocandin B0. Their isolation, structure elucidation and biological evaluation have been reported by Schmartz et al in “Cutaneous Antifungal Agents, 1993, pp 375-394”. The natural Echinocandin products are used as a precursor for semi-synthetic lipopeptides such as Anidulafungin, Micafungin, Caspofungin.
Anidulafungin is a semisynthetic lipopeptide Echinocandin B derivative developed by Eli Lilly/Versicor as an antifungal agent for intravenous administration. Anidulafungin is disclosed in U.S. Pat. Nos. 5,965,525 and 6,384,013, & are hereby incorporated by reference. Cilofungin is an Echinocandin lipopeptide disclosed by Eli Lilly in U.S. Pat. No. 4,293,489 for use as an antifungal agent, which is also hereby incorporated by reference. Micafungin (FUNGARD) is an Echinocandin-like lipopeptide developed by Fujisawa, as an antifungal agent for intravenous administration. Micafungin is disclosed in U.S. Pat. No. 6,107,458 which is also hereby incorporated by reference. Daptomycin (CIDECIN) is a semisynthetic lipopeptide derivative developed by Cubist as an antibacterial agent. Daptomycin is disclosed by Eli Lilly in U.S. Pat. No. 4,537,717 hereby incorporated by reference.
Pneumocandin B0 is produced as a secondary metabolite by fermentation of the fungus Glarea lozoyensis (U.S. Pat. Nos. 5,194,377 and 5,202,309) which is precursor for Caspofungin acetate (CANCIDAS), a semisynthetic lipopeptide Echinocandin B derivative currently being sold in US as an antifungal agent for intravenous administration.
The purification of Echinocandin lipopeptide compounds is a technical challenge despite intense research effort directed at controlling distribution and amount of various analogues produced by organism during fermentation. The organism can produce, in addition to desired compound, 20 or more Echinocandin-type compounds including Pneumocandin A0 and C0. Furthermore, these lipopeptides have unique solubility properties; they are essentially insoluble in water and most pure solvents, but dissolve in alcohols and some aqueous/organic solvent mixture. Moreover, it is difficult, if not impossible, to purify them by crystallization.
Therefore, the separation of any desired product like Pneumocandin B0 from its key analog impurities like pneumocandin C0, pneumocandin B5 and pneumocandin E0 is very cumbersome and expensive because all of them comprise a cyclic hexapeptide coupled with dimethylmyristate side chain.
Several efforts have been made to separate desired Pneumocandins from culture broth and to purify it by using various processes to obtain good productivity of highly purified Pneumocandins.
U.S. Pat. No. 4,874,843 describes the use of non-functional resin in reversed mode to purify Echinocandin-type products. Even though the process improved the purity of products derived from fermentation process, further improvements are still needed to remove contaminants that are difficult to separate from both the intermediate and final pharmaceutical compounds.
The use of SP-207, HP-20 resin and reverse phase HPLC are described for the separation of pneumocandin B0. (U.S. Pat. No. 5,162,211 and Robert E. Schwartz, The Journal of Antibiotics, Vol. 45, No. 12 1853-1866, 1992). R. E. Schwartz et. al. (1992) describe large scale whole broth methanol extract purification by use of fluidized bed of SP-207 followed by HP 20 column and then again use SP-207 for further material concentration and then purification of the same over silica column. This process has disadvantage that the purification method is very complicated because of being multi-step process. Furthermore, SP-207 and HP-20 are capital expensive to use at large scale production. Another disadvantage is that process is very time consuming and a major amount of desired product is lost in the transfer of sample from one column to another that decreases the yield of product of interest and makes the recovery of product poor.
U.S. Pat. No. 6,610,822 describes the purification process for Pneumocandin B0 where two phase, multi solvent system and back extraction steps have been used to maintain the polarity balance of the solution in which the compound is purified. The process comprises use of multiple solvents like isobutyl alcohol (IBA), methanol, n-heptane, water and acetonitrile, which is used in the extraction and purification of Pneumocandin B0. The problem associated with the said process is that recovery and reuse of multisolvent system involves critical monitoring as the process depends upon exact ratio of solvents to bring about polarity shift as described earlier. The process has low productivity, overall yield 69% and purity obtained is 61.4%.
US 2008/0108806 also describes purification of Echinocandin-type compounds such as pneumocandins, by two-phase system, first to remove non polar impurities and then extraction of compound followed by crystallization and precipitation. The first extraction is performed using non-polar or weakly polar water immiscible organic solvent followed by second extraction using water immiscible alcohols. The second extraction is followed by concentration and crystallization/precipitation. The crystallization/precipitation process by addition of antisolvent is facilitated using solid carriers. The process describes use of solid carriers to precipitate compound from the concentrated solution. This process also has low productivity.
U.S. Pat. Nos. 6,506,726 and 6,590,073 describe purification of echinocandin type compounds, especially of deacylated echinocandin B by chromatography.
EP1157030 describes formation of crystalline complex between echinocandin-type compounds and carbohydrate. The patent describes enhanced stability and water solubility for these complexes.
Since the potency of the final pharmaceutical product is dependent upon the purity of intermediates used to make the final product, therefore the improvements in purity at any stage of the manufacturing process is highly desirable. Ideally, the contaminants should be removed at the earliest stage possible in the manufacturing process.
Accordingly, conventional technologies of purifying pneumocandin B0 from the culture broth are problematic in that highly purified Pneumocandin B0 is not readily produced, stability of compound is not maintained, the recovery yield is relatively low and production cost are relatively high. Hence, there remains a long felt need to develop an improved purification process, which provides better resolution and productivity of Pneumocandins and other Echinocandin-type compounds at economical cost. Therefore, the object of the present invention is to develop an improved & economical purification process to obtain good yield and purity of certain Echinocandin-type compounds by using salt-adsorbent complex such as calcium phosphate. Moreover, the present process is simpler and provides improved productivity and purity than some of the existing process for the purification of Echinocandin-type compounds.
The present process is simple, cost effective, rapid, and scalable at industrial scale and provide highly purified yield of Echinocandin-type compounds at low cost as compared to prior art. The method allows for the removal of impurities by using economical salt-adsorbent complex and provide highly purified echinocandin type compounds which is highly improved in terms of purity than prior art and sufficient for further processing to obtain an active pharmaceutical ingredient such as, for example the antifungals caspogungin, anidulafungin, and micafungin.
Additionally this method reduces overall solvent and energy consumption making the process economically viable for commercial use and does not require chromatography steps involving expensive resins which are used in prior art processes for the isolation and purification of similar natural products.